REGENERATION OF BITTER GOURD (Momordica charantia L.) FROM LEAF SEGMENTS AND ROOT TIPS

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1 Progress. Agric. 18(2) : 1-9, 2007 ISSN REGENERATION OF BITTER GOURD (Momordica charantia L.) FROM LEAF SEGMENTS AND ROOT TIPS M. A. Z. Al Munsur, M. S. Haque, K. M. Nasiruddin and M. J. Hasan Department of Biotechnology, Bangladesh Agricultural University Mymensingh-2202, Bangladesh ABSTRACT Experiments were carried out to study the callus and subsequent plantlet regeneration ability of leaf segments and root tips of bitter gourd. A combination of 1.0 mg L -1 2,4-D and 1.0 mg L -1 BAP produced the highest percentage of callus in leaf segments (78.75%) and 1.0 mg L -1 2,4-D combined with 0.5 mg L -1 BAP produced the highest callus frequency in root tips (72.5%). Using the combination of BAP at 2.0 mg L -1 and NAA at 0.3 mg L -1 improved the callus frequency (80.00%) in root tips. Root tips showed better response than leaf segments in callus. Leaf segments and root tips had equal percentage (65.0) of shoot regeneration upon culture of the calli on medium with 2.0 or 2.5 mg L -1 BAP with 0.2 mg L -1 IAA. The highest length (3.95 cm) of shoot from leaf segments was recorded with 2.5 mg L -1 BAP, 0.5 mg L -1 IBA and 0.2 mg L -1 GA 3. The highest number of roots (6.75) and root length (2.45 cm) were found in 3.0 mg L -1 BAP with 0.1 mg L -1 NAA in root tips. The callus formation and regeneration ability of the explants have the potential for use in future biochemical studies and genetic improvement of bitter gourd. Key Words : Bitter gourd, In vitro regeneration, Leaf segments, Momordica charantia, Root tips INTRODUCTION Momordica is a genus of herbaceous annual or perennial climbers with 45 species native to tropical Asia and Africa. Momordica charantia is the most common among the six species cultivated for their fruits used as vegetables. It is believed to have originated in the tropics of the old world and is widely distributed in China, Malaysia, India, Tropical Africa and America (Kirtikar and Basu, 1994). Bitter gourd (Momordica charantia L.) belongs to the family Cucurbiteceae and is an economically important, expensive summer vegetable in Bangladesh. The fruits of bitter gourd contain nutritionally useful quantities of minerals and amino acids. The fruit contains very high amount of vitamins A and C, iron and minerals. Slices of unripe fruits are served as different types of curries and fried forms. The bitter taste is liked by some people and supposed to contain medicinal properties (Choudhury, 1967). It has hypoglycemic activity which reduces the blood glucose in diabetic rats (Perl, 1988; Khan, 1999). It has possible anti tumor activity (Xue et al., 1998), antispermatogenic and androgenic activities (Naseem et al., 1998). The abortifacient protein includes momorcharin from M. charantia. Bitter gourd has bright red seeds due to high lycopene content which can be used as an artificial food colorant (Yen

2 2 Regeneration of bitter gourd and Hwang, 1985). Bitter gourd protein (MRK29) has been reported to be used as HIV inhibitor (Jiratchariyakul et al., 2001). Bitter gourd is tolerant to a range of environments (Lim, 1998) and can be grown in tropical and subtropical climates (Reyes et al., 1994). Improvement of this crop and development of a new variety with desirable traits are obviously necessary which could be done through the applications of modern techniques of biotechnology. Genetic transformation techniques have been used as efficient tools for the breeding of crops. A reproducible protocol for in vitro regeneration is a prerequisite for varietal improvement through genetic engineering. Many factors determine in vitro regeneration. A balance between auxin and cytokinin regulates the in vitro regeneration from explants culture on artificial medium. The growth condition and suitable explants are also needed to be standardized for efficient utilization of tissue culture for genetic improvement of bitter gourd. There have not been many studies for the development of in vitro protocol to be used the improvement of bitter gourd in Bangladesh or its neighboring countries. The present study was, therefore, carried out to select the most suitable explants for in vitro regeneration, and to develop an efficient protocol for in vitro plant regeneration of bitter gourd. MATERIALS AND METHODS Seeds of a hybrid variety of bitter gourd marketed by the East West Seed (Bangladesh) Ltd. were purchased from local market. For the improvement of germination, the collected seeds were treated with hot water at C for 15 min by using the Vegetable Seed Treating Device developed by IPM Laboratory, Department of Plant Pathology, Bangladesh Agricultural University. Then the seeds were dried overnight under shade. The seeds were surface sterilized with 70% ethanol for 30 seconds and again then with 0.1% (w/v) mercuric chloride solution for five minutes. The seeds were then rinsed for 3-4 times by sterile distilled water to remove traces of HgCl 2. Then the seeds were placed on hormone free half strength MS (Murashige and Skoog, 1962) medium for germination. Seeds started germination within four days of inoculation and subsequently developed into rooted plantlets within 10 to12 days. Leaf segments and root tips were dissected out from the 12-days old plantlets and used as explants. The explants were then cultured on MS medium supplemented with various concentrations and combinations of 2,4-D (0.0, 0.25, 0.50, 1.0 and 5.0 mg L -1 ), BAP (0.2, 0.5, 1.0, 2.0, 2.5 and 3.0 mg L -1 ) and NAA (0.1, 0.3 and 0.6 mg L -1 ) for callus. For shoot regeneration, calli derived from both types of explants were cultured on MS medium supplemented with various concentrations and combinations of 2,4-D, BAP, NAA, IAA (0.1, 0.2, 0.6 mg L -1 ), IBA (0.5 mg L -1 ) and GA 3 (0.1 and 0.2 mg L -1 ). The ph of the medium was adjusted to 5.8 ± 0.1 before addition of 0.8% (w/v) agar for solidification. All the cultures were maintained in 16 h photoperiod, with light intensity of lux at 25 ± 1 C. Subcultures were carried out at regular intervals. Data on percentage of callus, days to callus initiation, percentage of shoot regeneration, days to shoot initiation, length of shoot, number of roots plantlet -1, days to

3 Munsur et al. 3 root initiation and length of roots were recorded. The experiments were arranged in Completely Randomized Design (CRD) with 4 replications. Data recorded for different parameters under study were statistically analyzed. The difference between the pair of means was evaluated at 5% level of significance by Duncan's Multiple Range Test (Gomez and Gomez, 1984). RESULTS AND DISCUSSION Effect of 2,4-D and BAP on callus Result revealed that leaf segments cultured on medium supplemented with 1.0 mg L -1 2,4-D combined with 1.0 mg L -1 BAP had produced friable and greenish calli (Fig. 1a). The highest callus frequency (78.75%) was found in 1.0 mg L -1 2,4-D combined with 1.0 mg L -1 BAP and was induced within the minimum number (10.75) of days (Table 1). The lowest (15.0%) percentage of callus was recorded in leaf segments cultured on medium with 0.20 mg L -1 BAP without 2,4-D. It can be noted that the frequency of callus increased when BAP and 2,4-D were used together (78.75 %) instead of single use of either of them (2,4-D, 58.75% and BAP, 38.00%). In root tip explants, 1.0 mg L -1 2,4-D combined with 0.50 mg L -1 BAP induced the highest frequency of callus (72.50%) within days and the lowest callus frequency (2.50%) was found in medium with 0.25 mg L -1 2,4- D and 0.50 mg L -1 BAP and it required relatively longer duration (40.25 days). Result also revealed that some combinations failed to produce any callus (Table 1). Both auxin and cytokinin applied separately were found to induce callus from the leaf of bitter gourd. However, a combined application of auxin and cytokinin was more efficient. Similar findings were reported earlier. Many workers agreed with this agreement in different crops. Josekutty et al. (1993) observed callus from leaf explants of Coccinia indica on MS medium supplemented with 1.5 mg L -1 BA, 0.5 mg L -1 Kn and 0.5 mg L -1 NAA. Kumar et al. (2003) found optimal embryogenic calli/embryos of Cucumis sativus L induced on B 5 medium supplemented with 2.0 mg L -1 2,4-D and 1.0 mg L -1 BAP. Effect of BAP and NAA on callus of bitter gourd from different explants Leaf segments cultured on medium supplemented with 2.0 mg L -1 BAP combined with 0.30 mg L -1 NAA produced the highest percentage of callus (68.75%) within the minimum number of days (9.75) (Table 2). The lowest frequency of callus (26.25%) was produced in a combination of 2.5 mg L -1 BAP and 0.3 or 0.6 mg L -1 NAA. Nabi et al. (2002) found that the combination of 1 mg L -1 BAP and 0.1 mg L -1 NAA was found suitable for callus from leaf explant of kakrol. Chaudhury et al. (2004) reported callus formation from leaf discs of tomato on 2.0 mg L -1 BAP and 2.0 mg L -1 NAA. In root tips, results of the present study revealed that 2.0 mg L -1 BAP combined with 0.3 mg L -1 NAA produced the highest callus (80.0%) which is statistically similar with 2.0 mg L -1 BAP combined with 0.10 mg L -1 (77.5) (Table 2). The lowest percentage of callus (27.50%) was produced in 3.0 mg L -1 BAP with 0.3 mg L -1 NAA and maximum number of days (33.25) was required for callus. Most of the calli produced from leaf segments were creamy and yellowish in colour (Fig. 1).

4 4 Regeneration of bitter gourd Fig. 1. Callus induced from different explants of bitter gourd cultured on media containing different concentrations of growth regulators : a) Leaf segments at 1.0 mg L -1 2,4-D and 1.0 mg L -1 BAP (18 DAC), b) Root tips at 1.0 mg L -1 2,4-D and 1.0 mg L -1 BAP (50 DAC), c) Leaf segments at 2.0 mg L -1 BAP and 0.3 mg L -1 NAA (16 DAC), d) Root tips at 2.0 mg L -1 BAP and 0.3 mg L -1 NAA (32 DAC) Effect of different concentrations of growth regulators on shoot regeneration In Leaf segments, the highest percentage (65.0%) of shoots formation was found in 2.5 mg L -1 BAP and 0.2 mg L -1 IAA and the lowest percentage (55.0%) was found in 2.0 mg L -1 BAP, 0.5 mg L -1 IBA and 0.2 mg L -1 GA 3 (Table 3). Costa et al. (2000) found higher shoot regeneration from cotyledon of tomato in 2.5 mg L -1 BAP combined with 0.2 mg L -1 IAA. Chen et al. (2000) also observed that shoot rate was highest on medium supplemented with 2.0 mg L -1 BA and IAA 0.02 mg L -1. The highest length of shoot (3.95 cm) was recorded in 2.0 mg L -1 BAP, 0.5 mg L -1 IBA and 0.2 mg L -1 GA 3 and lowest shoot length (3.23 cm) was found in 2.0 mg L -1 BAP and 0.2 mg L -1 IAA (Table 3). The highest percentage (65.0%) of regenerated shoots was found in 2.0 mg L -1 BAP and 0.2 mg L -1 IAA and the lowest (40.0%) was obtained in 1.0 mg L -1 BAP, 0.5 mg L -1 IBA and 0.2 mg L -1 GA 3 in root tips (Table 3). This result agreed with Shafiullah et al. (2003). They also observed highest number of shoots in 2.5 mg L -1 in combination with 0.2 mg L -1 IAA. The highest length of shoot (3.80 cm) was recorded in 2.0 mg L -1 BAP, 0.5 mg L -1 IBA and mg L GA 3) where the lowest (3.25 cm) was found in 2.5 mg L -1 BAP and 0.2 mg L -1

5 Munsur et al. 5 IAA. Different combinations of BAP, 2,4-D and NAA (Stated in materials methods) used in this experiment was unable to produce any plant. Shoots formed from root tips were healthier than those from root segments (Figs. 2a & b). Table 1. Effect of different combinations of 2,4-D and BAP on callus from leaf and root explants Concentrations (mg L -1 ) Leaf segments Root tips 2,4-D BAP % callus Days to callus % callus Days to callus k 0.00 h 0.00 k 0.00 d k 0.00 h 0.00 k 0.00 d j b 0.00 k 0.00 d i cd 0.00 k 0.00 d hi ef 0.00 k 0.00 d de cd 2.50 j ab d f i ab g ef i a h bc h abc f f ef abc f f de abc de cd fg abc c cd d abc b f a abc a g b abc ef de c c k 0.00 h b abc k 0.00 h b abc j a de abc k 0.00 h g bc In a column, the figures with the same letter (s) do not differ significantly; Twenty explants were cultured per treatment Effect of different concentrations of growth regulators on rooting of shoot The highest percentage (65.0%) of roots initiation was found in 2.5 mg L -1 BAP and 0.60 mg L -1 NAA and the lowest percentage (37.5) was found in 2.0 mg L -1 BAP and 0.2 mg L -1 IAA in leaf segments. The highest number of root (6.75) and length of root was found in 3.0 mg L -1 BAP and 0.1 mg L -1 NAA and lowest number (4.25) and length was found in 2.0 mg L -1 BAP and 0.2 mg L -1 IAA (Table 4). Agarwal and Kamal (2004) observed root differentiation of M. charantia on MS medium containing BAP and NAA. Ren et al. (2000)

6 6 Regeneration of bitter gourd also reported rooting of water melon to be achieved easily in half-strength MS medium and 0.1 mg kinetin or 0.1 mg NAA or 0.1 mg L -1 IAA. Table 2. Effect of different combinations of BAP and NAA for callus from leaf segments Concentrations (mg L -1 ) Leaf segments Root tips BAP NAA % callus Days to callus % callus Days to callus c a 0.00 f 0.00 f d c 0.00 f 0.00 f e c 0.00 f 0.00 f b e a e a 9.75 e a 24.50e d c bc d d d b d e c bc d e b c c f 0.00 f d b f 0.00 f e a f 0.00 f 0.00 f 0.00 f In a column, the figures with the same letter(s) do not differ significantly, Twenty explants were cultured per treatment Table 3. Effect of different concentrations and combinations of growth regulators for different parameters of shoot Growth regulators (mg L -1 ) Leaf segments Root tips Cytokinins (BAP) Auxins Percentage of shoots Shoot length Percentage of shoots Shoot length IAA c a e e a c e b 0.0 IBA GA b e d d e b 3.8 In a column, the figures with the same letter(s) do not differ significantly

7 Munsur et al. 7 In root tips, the highest percentage (70.0%) of root was found in 2.5 mg L -1 BAP and 0.5 mg L -1 IBA and the lowest percentage (37.5%) was found in 2.0 mg L -1 BAP and 0.2 mg L -1 IAA (Table 4). The highest number of roots (7.0) was found in 2.0 mg L -1 BAP and 0.5 mg L -1 IBA and the lowest (4.25) was found in 2.0 mg L -1 BAP and 0.2 mg L -1 IAA and 2.5 mg L -1 BAP and 0.2 mg L -1 IAA. The highest root length (2.25 cm) was found in 2.0 mg L -1 BAP and 0.3 mg L -1 NAA and 2.5 mg L -1 BAP and 0.6 mg L -1 NAA and lowest was found in 2.0 mg L -1 BAP and 0.2 mg L -1 IAA (Table 4). Fig. 2c shows a rooted plantlet of bitter gourd regenerated from root tip callus. Table 4. Effect of different concentrations and combinations of growth regulators for different parameters of root Growth regulators (mg L -1 ) Cytokinins (BAP) Auxins Percentage of roots * 2,4-D Leaf segments Number of root Root length (cm) Percentage of roots * Root tips Number of roots Root length (cm) b 5.50de g 0.00e cd 5.25e g 0.00e g 0.00g g 0.00e 0.00 IBA cd 6.25bc g 0.00e 0.00g b 6.50ab b 6.25b g 0.00g a 7.0a IAA f 4.25f f 4.25 d g 0.00g g 0.00e e 4.25f e 4.25 d g 0.00g g 0.00e g 0.00g g 0.00e g 0.00g g 0.00e 0.00 NAA g 0.00g d 5.38 c ab 6.30abc b 6.30b c 6.25bc g 0.00e a 5.95cd c 4.95 c d 6.75a e 4.75 c e 5.25e g 0.00e 0.00 In a column, the figures with the same letter(s) do not differ significantly, * Roots formed either regenerated shoot or callus

8 8 Regeneration of bitter gourd Fig. 2. a) Shoot regeneration from root tip callus of bitter gourd cultured on media containing 2.0 mg L -1 2,4-D, 0.50 mg L -1 IBA and 0.20 mg L -1 GA 3; b) Leaf segments callus cultured on media containing at 2.5 mg L -1 BAP and 0.20 IAA mg L -1 (35 DAC); c) Rooted plantlets from root tip callus cultured on media containing 2.0 mg L -1 BAP, 0.50 mg L -1 IBA and 0.20 mg L -1 GA 3 (80 DAC) Establishment of plants After sufficient development of shoot and root systems, the small plantlets were taken out from the culture vessels with proper care without damaging the roots. The plantlets were then transplanted in small pots. When the plantlet grew to a height of 5-6 cm, they were transferred to plastic pots. The growth condition of plantlets was satisfactory and the survivability of plant more than 75%. The pots with the established plants were then kept in natural condition, allowed them to grow with proper care. ACKNOWLEDGEMENT The research grant from Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka. is gratefully acknowledged. REFERENCES Chaudhury, Z., Habib, D., Rashid, H. and Qureshi, A. S Regeneration from various explants of in vitro seedling of tomato. Pakistan J. Biol. Sci., 7(2) : Chen, H., Zhang, J., Zhong, J. and Yu, J Plant regeneration from hypocotyl explants cultured in vitro and histological observations in Lycoperiscon esculentum Mill. Xibei Zhiwu Xuebao, 20(5) : Choudhury, B Vegetables National Book Trust. New Delhi, India. Costa, G. M., Noqueira, F. T. S., Otoni, W. C. and Brommonschenkel, S. H In vitro regeneration of processing tomato (Lycopersicon esculentum Mill.) IPA-5 and IPA-6. Cienciae-Agrotecnologia, 24(3) : Gomez, A. C. and Gomez, A. A Statistical Procedures for Agricultural Research (2 nd edn.). John Wiley and Sons, New York. pp c

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